Orthogonal analytical approaches to detect potential contaminants in heparin

Abstract

Heparin is a widely used anticoagulant and antithrombotic agent. Recently, a contaminant, oversulfated chondroitin sulfate (OSCS), was discovered within heparin preparations. The presence of OSCS within heparin likely led to clinical manifestations, most prevalently, hypotension and abdominal pain leading to the deaths of several dozens of patients. Given the biological effects of OSCS, one continuing item of concern is the ability for existing methods to identify other persulfonated polysaccharide compounds that would also have anticoagulant activity and would likely elicit a similar activation of the contact system. To complete a more extensive analysis of the ability for NMR and capillary electrophoresis (CE) to capture a broader array of potential contaminants within heparin, we completed a systematic study of NMR, both mono-and bidimensional, and CE to detect both various components of sidestream heparin and their persulfonated derivatives. We show that given the complexity of heparin samples, and the requirement to ensure their purity and safety, use of orthogonal analytical techniques is effective at detecting an array of potential contaminants that could be present.anticoagulant ͉ capillary electrophoresis ͉ contamination ͉ NMR ͉ oversulfated chondroitin

Chemically Oversulfated Glycosaminoglycans Are Potent Modulators of Contact System Activation and Different Cell Signaling Pathways

“…S2B). Consistent with our published observations that the heparins are not contaminated with OSCS alone (7,8); the published CE data (6,34) also showed that an OSHS co-migrated with heparin (see Ref. 6, supplemental Fig.…”

“…One peak was named fully sulfated CS and the other was named partially sulfated OSCS in the published report (34). In addition, we found that less sulfated DS and HS (OSDS-M, OSDS-L and OSHS-M, OSHS-L) had several proton signal peaks such as 2.04, 2.07, 2.10, and 2.12 ppm for OSHS-M and OSHS-L and/or OSDS-M and OSDS-L, which made the 2.07 Ϯ 0.02 and 2.12 Ϯ 0.02 ppm proton chemical shifts not specific for DS and OSDS, respectively, in contrast to the published reports (6,34). Because the heparin contaminant that migrated before heparin, based on CE analysis, was defined as OSCS or heparin contaminant during the past 2 years, we decided to choose all the OSGAGs that migrated like OSCS (Fig.…”

“…The less sulfated GAGs are removed from crude heparin into two heparin by-products named GAG waste and tank bottom (6). We have analyzed the same contaminated heparin preparations and concluded that heparin is contaminated either with chemically oversulfated/desulfated GAG waste or chemically oversulfated/desulfated GAG waste plus oversulfated tank bottom (7,8).…”

Polyacrylamide Gel Electrophoresis

“…3). Structural integrity of gel-eluted oligosaccharides was assessed by two-dimensional correlation spectroscopy (2D COSY) and 2D heteronuclear multiple quantum coherence (2D HMQC) spectroscopy [20]. Comparison of the 2D COSY and 2D HMQC spectra of the BLH oligosaccharide mixture and gel-eluted oligosaccharides confirmed that the saccharide backbone structure remains unchanged after the CE-PAGE (Fig.…”

Identification of Chemically Sulfated/desulfated Glycosaminoglycans in Contaminated Heparins and Development of a Simple Assay for the Detection of Most Contaminants in Heparin

“…5 The less sulfated GAGs are removed from crude heparin during pharmaceutical grade heparin production into two heparin byproducts named GAG waste and tank bottom. 6 GAG waste has a NMR profile highly resembles heparin 6 but it consists of less sulfated heparin/heparan sulfate and dermatan sulfate. The tank bottom consists of even less sulfated heparan sulfate, chondroitin sulfate, and dermatan sulfate.…”